Commercial production of protein therapeutics and other biological products such as monoclonal antibodies is presently carried out generally in bioreactors adapted for culturing suspensions of genetically optimized mammalian, insect or other cell types. Mammalian cell culture bioreactors typically have several hundred to several thousand liters in working volume. Most common full scale manufacturing plants have bioreactors with working volumes ranging from approximately 1,000 liters up to 25,000 liters. Drug candidates for clinical trials are produced in laboratory scale bioreactors having five (5) liters to several hundred liters of working volume.
The optimization to achieve the highest biological product yields possible in the smallest amount of time and the related challenges of bioreactor scale-up have focused on the control of recognized critical process parameters such as pH, dissolved oxygen (DO), temperature, nutrient composition and by-product profiles, agitation profile, gas sparging method, nutrient feed and product harvest profiles. The importance of other process parameters such as dissolved carbon dioxide (dCO2) and osmolality (i.e. concentration of dissolved particles per kilogram of solution) is just recently being documented in the literature. As a matter of fact, many commercial bioreactors do not even have the means installed to measure dissolved carbon dioxide and/or osmolality levels in-situ, let alone a means to control and optimize those parameters. Depending on the scale of the commercial operation—ranging from hundreds up to 25,000 liters of bioreactor volume—scale-up, optimization and control of the process pose different challenges. At commercial scales above about 1,000 liters, simultaneous and independent control of dissolved carbon dioxide and osmolality levels becomes difficult if not impossible with current best available technologies and methodologies.
Before a manufacturing-scale mammalian cell cultivation process starts in a bioreactor, a seed culture inoculum is typically prepared. This involves culturing production cells in a series of flasks in incubators and/or smaller bioreactors of increasing volume until enough cells are available for inoculation into the production bioreactor. The process involves transferring a cell population from one culture vessel to a larger one. Generally, a 20% dilution of the cell population is used for each transfer or subculture. In the incubator, the flasks with culture medium are clamped to a rotating platform to swirl the culture and facilitate gas transfer between the culture medium and the atmosphere in the incubators. Typically, the incubator for a mammalian cell culture process is set at 37° C. with 5% carbon dioxide (CO2) and a humidity level higher than about 80%. Similar temperatures and CO2 levels are used for seed cultures grown in bioreactors. When the seed culture reaches a sufficient volume and cell density, it is inoculated into the production bioreactor.
After seed culture is inoculated into the bioreactor medium, parameters such as pH, temperature, and level of dissolved oxygen are controlled to the prescribed levels during the cell cultivation process. pH is typically controlled by adding basic or acidic solutions when necessary during the process. Commonly used base solutions include sodium bicarbonate, sodium carbonate and sodium hydroxide solutions. Dissolution of carbon dioxide (CO2) is commonly used to achieve a more acidic pH. The preferred temperature of the culture medium or solution for mammalian cell cultivation processes is about 37° C. The desired level of dissolved oxygen in the culture medium or solution is typically achieved through air sparging using sparger installed on the bottom of the bioreactor, along with agitation of the culture medium or solution using impellers which breakup the large air/oxygen bubbles to enhance the transfer of oxygen to the cell medium from the sparged air bubbles. Purging the bioreactor headspace with a cover gas provides a limited degree of surface gas exchange. Disadvantageously, air-sparging and agitation of the culture medium or solution may result in foaming and shear damage to the mammalian cells which adversely impacts cell viability. Accumulations of foam on the surface of the culture medium also serve to further limit surface gas exchange and to reduce the available working volume of the bioreactor.
Commercial-scale mammalian cell cultivation processes may be conducted in three different operation modes: batch mode or fed-batch mode for suspended cell cultures, and perfusion mode for immobilized cells. The majority of the commercial-scale mammalian cell cultivation processes are operated in fed-batch mode. In fed-batch mode, additional media and nutrients are added to the bioreactor at different times during the cell cultivation process to supplement the carbon source and other nutrients after initial bioreactor setup.
Before any bioreactor is used for mammalian cell cultivation, it typically must be sterilized and equipped with various probes as well as connections for supplemental gas supply and introduction of additional feeds. Temperature probes, pH detectors, dissolved oxygen probes and dissolved CO2 probes or sensors are used to monitor the temperature, pH, dissolved oxygen and dissolved CO2 levels of the cell medium or solution in real time. In addition, cell culture medium or solution samples can be withdrawn from the bioreactor at selected intervals to determine cell density and cell viability, as well as to analyze other characteristics such as metabolites and osmolality. Based on such analytical results, additional feed or other additives can be added to the cell culture medium or solution in an effort to prolong the cell viability and increase production of biological products. When cell viability reaches a prescribed lower threshold, the cell cultivation process can be stopped or shut down. The prescribed lower threshold is often determined empirically based on the results of down-stream recovery and purification of the harvested biological products.
During the cultivation process, the mammalian cells exhibit three phases, namely the lag phase, the exponential growth phase, and the stationary or production phase. The lag phase occurs immediately after inoculation and is generally a period of physiological adaptation of mammalian cells to the new environment. After the lag phase, the mammalian cells are considered in the exponential growth phase. In the exponential growth phase, the mammalian cells multiply and cell density increases exponentially with time. Many cells actually start to produce the desired protein, antibody or biological product during some point in the exponential growth phase. Cell density refers to the total number of cells in culture, usually indicated in the density of viable and non-viable cells. When the mammalian cells reach the stationary or production phase, the viable cells are actively producing the biological products for downstream harvesting. During this phase, the total cell density may remain generally constant, but the cell viability (i.e. the percentage of viable cells) tends to decrease rapidly over time.
Mammalian cells are known to be sensitive to the amount of dissolved carbon dioxide in the cell culture media or solution. Mammalian cell cultures exposed to excess carbon dioxide levels during the exponential growth phase may demonstrate reduced production of monoclonal antibodies or other desired biological products. Before inoculation, the pH of the slightly alkaline culture media is often reduced to a more optimal value by addition of carbon dioxide. This process often leads to elevated levels of dissolved carbon dioxide at the beginning of the lag phase of many mammalian cell culture processes.
Dissolved carbon dioxide in mammalian cell culture bioreactors originates from chemical and biological sources. The chemical source of carbon dioxide is equilibrium chemical reactions occurring within the cell culture medium or solution that includes a selected amount of a buffer solution containing sodium bicarbonate and/or sodium carbonate. Additionally, carbon dioxide may be directly sparged into the slightly alkaline culture medium or solution to reduce the pH level of the broth to a prescribed level, usually around 7.0, resulting in more dissolved carbon dioxide. The biological source of carbon dioxide is a product of the respiration of the mammalian cells within the bioreactor. This biological source of carbon dioxide increases with cell density and generally reaches its maximum value at about the same time that cell density within the bioreactor is maximized. However, as more carbon dioxide is produced, the pH of the cell culture medium trends toward acidic such that additional bicarbonate is needed to keep the pH of the cell culture medium or solution within the desired range.
To offset the effects of increased dissolved carbon dioxide, one may add sodium bicarbonate so as to maintain the pH of the solution within the prescribed range or attempt to strip the carbon dioxide from the solution by sparging with additional air. Both of these means to offset the effects of increased carbon dioxide have other negative consequences on the mammalian cell culture process.
First, adding sodium bicarbonate to adjust the pH of the solution, results in an increase in osmolality level. Osmolality level represents the number of dissolved particles per kilogram of solution and is commonly reported as mOsm/kg by freeze-point depression. It is known in the art that increased levels of either dissolved carbon dioxide or increased levels of osmolality have adverse or negative impacts on cell density or yield. However, the combined or synergistic effects of carbon dioxide and osmolality levels are not well understood.
Carbon dioxide dissociates into bicarbonate ions at a pH of 7 in water. Only a fraction of the carbon dioxide remains as free CO2 in an un-dissociated state. Removing the dissolved carbon dioxide from a cell culture thus becomes difficult as most mammalian cell cultures take place at pH levels in the range of 6.5 to 7.5. The dissociated bicarbonate ions are not easily removed and generally must be recombined into free carbon dioxide before they can be stripped out of the solution. Any addition of sodium bicarbonate to balance the pH will also increase the equilibrium dissolved carbon dioxide concentration or saturation level in the solution, making it more difficult to remove the carbon dioxide physically.
Conventional methods of removing or stripping dissolved carbon dioxide from a mammalian cell culture solution is by sparging the cell culture solution with air or a gas mixture of air/oxygen/nitrogen in agitated tanks. However, gas sparging in agitated tanks results in adverse effects to the cell culture process. In particular, the gas-bubble breakage at the tip of the rotating agitator is a source of high shear rate that damages mammalian cell membranes, often sufficiently to cause cell death. Even when damage is sub-lethal, cell productivity is compromised in the period that the damaged membrane is repaired. In most current bioreactors, the agitator is a radial flow type that rotates around the center axis of the reactor vessel, and where the sparged gas and liquid within the reactor vessel are pushed outwards from the center of the reactor vessel to the side wall of the vessel. The main purpose of radial pumping impellers is to break and disperse gas bubbles provided by spargers. Bubble breakage behind the rotating impeller will have a major role in cell death. Small shading vortices formed behind the impeller will also damage cells to a lesser extent. Such impellers impart very little vertical or axial mixing. If multiple radial impellers are used, they may form distinct mixing zones within the reactor vessel. Current commercial axial flow impeller designs are all downward pumping. Downward pumping axial impellers generate vortices that entrain gas from the headspace into the body of the agitated liquid, resulting in gas bubble formation. As mentioned above, gas bubbles have a negative impact on cell growth in that the force of a breaking gas bubble is sufficient to damage the outer membrane of a mammalian cell, and can cause it to burst. Therefore, conventional radial impellers and downward pumping axial impellers are not generally suitable for promoting gas exchange between the liquid surface and the bioreactor headspace as a way to remove carbon dioxide from the cell culture medium.
In commercial scale bioreactors (e.g. 1,000 liters to 25,000 liters), carbon dioxide removal is more difficult than in smaller reactors, and the excess carbon dioxide that tends to accumulate is detrimental to cell growth. During scale-up from a bench or laboratory scale bioreactor to a production or commercial scale bioreactor, a productivity loss of up to 60% has been observed; excessive levels of carbon dioxide at the larger scale is the suspected cause of such productivity loss. Carbon dioxide removal via air sparging tends to be very effective in laboratory or bench scale bioreactors (e.g. less than 10 Liters of working volume) but is less effective in larger scale commercial bioreactors for at least three reasons: (i) the surface area to volume ratio is reduced, which further limits surface gas exchange; (ii) higher hydrostatic pressures in a large vessel increase carbon dioxide solubility; and (iii) larger vessels contain more cells and the resulting increased need for sparged gas to supply oxygen leads to more bubbles, which create more foam at the surface and further inhibit surface gas exchange.
Another disadvantage of foam created by air sparging into a rotating agitator is that cells become trapped on the foam layer where they are depleted of nutrients. Foaming also limits the operable volume of the reactor, as foam overflow can damage the integrities of the biological filters that prevent process contamination. Although anti-foaming agents are used, such agents have many undesirable effects. For example, anti-foaming agents can contaminate the biological products and their removal may require further downstream purification steps. Also, many such anti-foaming agents reduce the interfacial gas-liquid mass transfer efficiencies occurring within the bioreactor.
Also, gas bubbles created by sparging can burst at the liquid surface; this is often more damaging to cultured cells than shear due to the agitator. Minimizing agitator speed and limiting the gas sparging rate are currently viewed as the best means to avoid such damage and increase cell viability. However, both measures reduce the amount of carbon dioxide that is removed which in turn inhibits cell growth and reduces viability. These disadvantages are particularly challenging to overcome in large, commercial-scale bioreactors where the shear rate goes up substantially with the diameter of the impellers.
Some bubble free systems with membrane aeration have been proposed, but these have demonstrated only limited success even at small scales. Membrane fouling, system cost and system scalability have prevented membrane-based bioreactors from gaining broader acceptance.
Wave Bioreactor™ is an example of a design in which the surface to volume ration is large enough for dissolved carbon dioxide to be removed through gas exchange at the surface. Agitation is provided by rocking motion of a mechanically supported tray (See FIG. 1). The surface area needed for sufficient gas exchange has limited the size of this bioreactor to less than 500 liter working volume which is not suitable for large, commercial scale systems.
Conical bioreactors have also been proposed as an alternative way to provide a surface area to volume ratio large enough for gas exchange. The conical bioreactor is supported on an orbital shaker that provides gentle rocking motions. Much like the Wave Bioreactor™, mechanical engineering issues limit this design to smaller bioreactors